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Comparative Study of Nanobioactive Glass Quaternary System 46S6 Mostafa Mabrouk, Amany Mostafa, Hassane Oudadesse, Eric Wers, Anita Lucas-Girot, Mohamed I. El-Gohary

To cite this version: Mostafa Mabrouk, Amany Mostafa, Hassane Oudadesse, Eric Wers, Anita Lucas-Girot, et al.. Comparative Study of Nanobioactive Glass Quaternary System 46S6. Bioceramics Development and Applications, 2014, 4 (1), pp.1000072. .

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ISSN: 2090-5025

Bioceramics Development and Applications

The International Open Access Bioceramics Development and Applications

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Mabrouk et al., Bioceram Dev Appl 2014, 4:1 http://dx.doi.org/10.4172/2090-5025.1000072

Bioceramics Development and Applications Research Article

Open Access

Comparative Study of Nanobioactive Glass Quaternary System 46S6 Mabrouk M1,2, Mostafa AA1,2*, Oudadesse H1, Wers E1, Lucas-Girot A1 and El-Gohary MI3 1

University of Rennes 1, UMR CNRS 6226, 263 av. du général leclerc, 35042 Rennes cedex, France Biomaterials Department, National Research Centre (NRC), Cairo, Egypt 3 Physics Department, Faculty of Science, Al -Azhar University, Cairo, Egypt 2

Abstract Different bioactive glass systems have been prepared by sol-gel. However, the production of Na2O-containing bioactive glasses by sol–gel methods has proved to be dificult as the sodium nitrate used in the preparation could be lost from the glass structure during iltration and washing. The aim of this study was to prepare the quaternary system 46S6 of bioactive glass by modiied sol-gel techniques with a decrease in the time of gelation. In addition, compare the behaviour of the prepared sol-gel bioactive glass system by its corresponding prepared by melting. The obtained glasses were characterized by using several physicochemical techniques; XRD, FTIR, TEM and SEM beside the effect of the glass particles on the viability of osteoblast like cells (Saos-2). Results show that nanopowders 40-60 nm of 46S6 glass system had been prepared by modiied sol-gel (acid-base reaction) method at 600°C in just three days at 600°C. Cell viability by MTT assay conirmed the effectiveness of the prepared nanobioactive glass.

Keywords: Nanoshperes; bioactive glass; sol-gel; melting; 46S6; Heat treatment and cell viability Introduction Among the production methods of bioactive materials stands out the sol-gel processing. he prepared materials with high purity, homogeneity and lower processing temperatures could possibly be obtained [1]. Commercially available glass compositions; 45S5, 52S4, 58S and 64S could be synthesized by sol-gel method [2-4]. Addition of some elements such as K2O and Na2O used in conventional melting, reduce the melting temperature and makes the inal materials are more soluble in aqueous media. his factor is extremely important for the interaction of the materials with the living tissues. However, to the best of knowledge nanobioactive glass 46S6 system containing high amount of Na2O has not been achieved yet. he particle size of the traditional sol–gel-derived bioactive glasses was bigger than 1 μm [5] because of the long gelation and ageing time [4,5]. he objective of this study was to prepare the quaternary system 46S6 of bioactive glass by modiied sol- gel techniques in shorter time as previously mentioned. In addition, comparing the behaviour of the prepared bioactive glass system by sol-gel named (SG-B) by its corresponding system prepared by melting technique named (MB) by means of XRF, XRD, FTIR, TEM and SEM before and ater immersion in simulated body luid (SBF). Furthermore, the viability test was conducted on the Saos-2 cells using MTT assay.

Synthesis and Characterizations Synthesis he 46S6 bioactive glass with the composition (46% SiO2, 24% CaO, 24% Na2O, 6% P2O5 wt%) was prepared by melting as previously reported [6]. he used chemicals were calcium silicate (Alfa Aesar), trisodium trimeta phosphate and sodium metasilicate pentahydrate (Sigma). he prepared glass by melting technique was named MB. However, the bioactive glass with the same composition was prepared by sol-gel technique and named SG-B. Tetraethyl orthosilicate (TEOS: Fluka, wt=208.33), 56 ml was added to 50% ethanol at room temperature. he pH was adjusted at 2 by nitric acid with continuous stirring for 1h. Addition of 24.5 g of calcium nitrate hydrate (Fluka, M.wt=236) and then, 21.07 g of sodium hydroxide (Prolab, M.wt=40) were added to the above mixture (the previous mixture was named solution A). Polyethylene glycol (PEG-Fluka M.wt=600) 10 g was Bioceram Dev Appl ISSN: 2090-5025 BDA, an open access journal

dissolved in distilled water at room temperature then, 3.43 g of ammonium dihydrogen phosphate (MERK, M.wt=115.03) was added to the PEG solution, this mixture was named solution B. Solution (B) was gradually added to solution (A) with continuous stirring over night in a Telon container. he resulted solution was iltered and washed with distilled water 3 times and then with ethanol by using centrifuge at 1650 rpm for 10 min each. Drying of the washed gel at 70°C overnight and then calcination at 600°C for 2 h have been performed.

Characterizations he thermal analysis of both MB and SG-B powders were investigated by DSC/TG. he structural and morphological properties were characterized by using XRF, XRD, FTIR, TEM and SEM. For evaluation of the in-vitro bioactivity, 100 mg of powders were immersed in 200 ml of SBF at diferent times (2, 5 and 7 days). Furthermore, the granules were iltered, cleaned with ethanol, and dried at 60°C overnight. he Physicochemical properties of the iltered granules were studied by XRD, FTIR and SEM. Cell viability test was conducted on Saos-2 cells to evaluate the biocompatibility of the prepared bioactive glasses materials by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT) assay.

Results and Discussion DSC/TG analysis was employed to determine the thermal characteristics of the investigated powder samples as in Figure 1. For the prepared powder samples MB and SG-B, three characteristics peaks at 559°C, 727.6°C and 1235°C and at 550.6°C, 605.5°C and 835.8°C corresponding to the temperature of vitreous transition

*Corresponding author: AA Mostafa, University of Rennes 1, UMR CNRS 6226, 263 av. du général leclerc, 35042 Rennes cedex, France, Biomaterials Department, National Research Centre (NRC), Cairo, Egypt, Tel: +20100 6600 620 ; E-mail: [email protected]; [email protected] Received Decenber 31, 2013; Accepted January 05, 2014; Published January 12, 2014 Citation: Mabrouk M, Mostafa AA, Oudadesse H, Wers E, Lucas-Girot A, et al. (2014) Comparative Study of Nanobioactive Glass Quaternary System 46S6. Bioceram Dev Appl 4: 072. doi: 10.4172/2090-5025.1000072 Copyright: © 2014 Mabrouk M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Citation: Mabrouk M, Mostafa AA, Oudadesse H, Wers E, Lucas-Girot A, et al. (2014) Comparative Study of Nanobioactive Glass Quaternary System 46S6. Bioceram Dev Appl 4: 072. doi: 10.4172/2090-5025.1000072

Page 2 of 4 (Tg), crystallization temperature (Tc) and temperature of fusion (Tf), respectively have been presented. he thermal stability is the stability of a molecule at high temperatures; i.e. a molecule with more stability has more resistance to decomposition at high temperatures. It corresponds to (Tc–Tg) and equal to 168°C and 54.9οC for MB and SGB, respectively. his means that sol-gel method induces decreasing of 46S6 bioactive glass thermal stability than melting method. Variation in thermal stability afects on the chemical stability of the prepared glasses and suggests higher bioactivity [7]. Figure 2 shows the XRD patterns of the 46S6 bioactive glass prepared by melting technique (MB) at 1350°C and by sol-gel method at diferent heating temperatures from 70°C overnight to 600°C/2 h. he characteristic peak of MB presents a difraction halo between 20° and 37° (2θ) with centre at 32°C. However, SG-B shows this halo without others at 600°C during 2h i.e. above the Tg (550°C) and below Tc (605.5°C) as conirmed by DSC/TG results and as reported for the XRD of silicate glass [1]. Figure 3 shows the FTIR spectra of the MB and SG-B at 600°C/2h. he spectrum of both samples indicates the same chemical structure as the inger print region is almost the same. It presents the characteristic of silicate absorption bands at 467, 945 cm−1 and that of phosphate groups at 600, 740 and 1045 cm−1 as reported [1,7,8]. A band at 1653 cm-1 attributed to C-O resonance in CO32- group is detected and another

at 3460 cm−1 characteristic to Si-OH is clearly observed for sample SGB. [9]. Certainly, it is clear that using of diferent preparation methods for the same chemical composition does not afect on the nature of chemical bonds between atoms. he quantitative analysis of the prepared powder samples MB and SG-B were determined by XRF. he chemical composition of MB and SG-B was presented in Table 1. It is obvious that the two samples have the same chemical composition with small fractions of impurities (0.892% for MB and 0.406% for SGB). his means that SG-B has less impurities than MB. herefore, this conirms that the prepared 46S6 bioactive glass by sol-gel method has a high purity and considers as good for cell viability [1]. TEM is a powerful tool for observing the morphology and size of particles. Figure 4 shows the TEM micrographs of the prepared samples, MB shows agglomerated sphere particles (Figure 4a) however, SG-B shows highly homogenous nano-spheres ranging between 40-60 nm (Figure 4b). hese diferences in the homogeneity and particle size are referring to the preparation methods. One-step acid catalysis bioactive glasses require long gelation times. his allows for the aggregation and growth of colloidal particles in the solution, leading to inal products with microscale particle sizes [10]. However, in this work, two-step; acid–base catalysis was followed. he addition of sodium hydroxide, as a second catalyst, to the sol that was initially catalyzed by nitric acid was found to raise the rate of condensation and decrease gelation time to few hours. he condensation rate is proportional to [OH-] above the isoelectric point. In this study, sodium hydroxide was used as source of Na2O and for gelation to provide an environment of a pH much higher than the isoelectric point of silica [11]. Figure 5 shows the XRD patterns for MB (Figure 5a) and SG-B (Figure 5b) before and ater immersion in SBF at diferent time intervals (2, 5 and 7 days) in reference to the pattern of synthetic hydroxyapatite. Ater two days of immersion in SBF solution, two characteristic amorphous peaks of hydroxyapatite (HA) at 25.80° and 31.79° are observed for the sample SG-B (Figure 5b). he intensity and degree of crystallinity of these peaks increases with the increasing of the

MB

SG-B Si-O-Si bending

FT-IR

Si-oH

Figure 1: DSC /TG for MB and SG-B.

PO+SiO2 SiO2 PO C=O SB 600°C/2h

MB 4000 3500 3000 2500 2000 1500 1000 500

SG-B 600°C /2h

wavenumber cm-1 Figure 3: FTIR for MB and SG-B.

Intensity (counts)

SG-B 500°C /2h SG-B 400°C /2h

SG-B 300°C /2h

a

b

SG-B 200°C /2h SG-B 70°C overnight

10

20

30

2 θ 40

MB 50

60

70

Figure 2: XRD of the prepared bioactive glasses at different treating temperature.

Bioceram Dev Appl ISSN: 2090-5025 BDA, an open access journal

Figure 4: TEM image of a. MB b. SG-B.

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Page 3 of 4 soaking time as it is clearly observed with high crystallinity ater ive and seven days of immersion. It is also notable the appearance of new peak at 45.142° with higher intensity than that observed in synthetic HA. his could be due to the over lapping of this peak with the peak of Rhombohedral calcium phosphate with that appears at 56.462° as previously reported [7]. On the other hand for MB ater two and ive days of immersion we can't observe any characteristic peaks for HA, but ater seven days two characteristic peaks for HA at 25.80° and 31.79 could be observed. his conirms the high reactivity of the glass sample prepared by sol-gel than that prepared by melting. Figure 6 shows the FTIR for samples MB and SG-B before and ater

immersion in SBF at diferent periods. It is clearly evident almost the same spectra. However, ater two days of immersion a band at 945 cm-1 of SiO2 for sample MB could be recognized, while, it is hardly observed for SG-B ater the same period of immersion. Ater 7 days, the spectrum is quite similar to that of hydroxyapatite except two bands located at 1620 and 3423 cm-1. hese absorptions bands are characteristic of the presence of water related to the hygroscopic feature of the formed apatite. he OH band at 3561 cm-1 is included in the H–O–H band at 3423 cm-1. he high water content is probably due to the presence of strong nucleophilic groups such as: P–OH or Ca–OH, which favor the adsorption of the ambient humidity [7]. Also, it could be noticed that the intensity increase for bands at 600 and 740 cm-1 which is characteristic to PO43- and PO assigned to crystalline calcium phosphate. However, for MB there are no notable changes for this band. his conirms the formation of HA layer on the surface of SG-B more faster than on MB as proved by XRD results. Figure 7 shows the SEM micrographs for samples MB and SG-B before and ater immersion in SBF at diferent time intervals. For both samples images MB and SG-B before immersion a homogeneous amorphous bulk with almost uniform particles size has been observed. Ater immersion in SBF for two days there is no notable precipitation can be seen for MB but on the other hand for SG-B it could be noticed a layer of precipitated HA, which accumulate with the time to form a multi-layers of HA as it is evident ater seven days. his is in agreement with the results of XRD and FTIR [1]. Cell viability was tested by MTT assays and is presented as histogram in Figure 8. he cells treated with diferent concentrations of the prepared samples showed relatively good cell viability compared to the control used as reference. he prepared bioactive glass by solgel method SG-B shows a higher tendency for cell growth at higher concentrations than those made by melting technique owing to their higher reactivity due to its smaller particle size and higher surface area resulted from shortened gelation time and thermal stability diference [1,9,10,11].

Figure 5: XRD for samples a. MB and b. SG-B before and after immersion in SBF for 2, 5and 7 days.

a

C=OC–O

O-H

Si-O-Si bending

PO+SiO2 SiO2

FTIR %

MB After 7 days

MB After 5 days

MB After 2 days

MB before immersion 4000

b

3500

3000

2500

2000

wavenumber CM-1

C=O

O-H

1500

C–O

1000

500

Si-O-Si bending

PO+SiO2

PO PO4-3

FTIR

SG-B after 7 days SG-B after 5 days

SG-B after 2 days

SG-B before immerssion 4000

3500

3000

2500

2000

1500

Wavenumbers Cm-1

1000

500

Figure 6: FTIR for samples a. MB and b. SG-B before and after immersion in SBF for 2, 5and 7 days.


Figure 7: SEM micrographs; a, c, e and g for sample MB before and after immersion in SBF for 2,5, 7 days and b, d, f and h for sample SG-B before and after immersion in SBF for 2,5, 7 days, respectively.

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Page 4 of 4 Acknowledgment 450 400

MB

% of control

350

305

300

This work was inancially supported by Campus France and Academy of Scientiic Research and Technology (ASRT) of Egypt, Imhotep program.

377

SG-B 325

314

References

250 256

1. Pereira MM, Clarck AE, Hench LL (1994) Journal of Materials, Synthesis and Processing 2: 189-195.

250 200 150

2. Sepulveda P, Jones JR, Hench LL (2001) Characterization of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses. J Biomed Mater Res 58: 734-740.

100100

100

3. Theodorou G, Goudouri OM, Kontonasaki E (2011) Comparative Bioactivity Study of 45S5 and 58S Bioglasses in Organic and Inorganic Environment. Bioceramics Development and Applications 1:1-4.

50 0 0 300 600 SG-B and MB concentraion(µg /ml)

1200

4. Mezahi FZ, Lucas-Girot A, Oudadesse H, Harabi A (2013) Reactivity kinetics of 52S4 glass in the quaternary system SiO2-CaO-Na2O-P2O5: Inluence of the synthesis process: Melting versus sol-gel. J Non-Cryst. Solids 361: 111-18.

Figure 8: MTT assay of MB and SG-B after 48 hour of exposure.

Sample Name

SiO2%

CaO%

Na2O%

P2O5%

SG-B

47.49

24.8

20.58

6.73

MB

44.04

27.71

20.62

6.31

Table 1: Chemical analysis of MB and SG-b by XRF.

Conclusions Nanobioactive quaternary glass system 46S6 has been prepared by modiied sol-gel (acid-base reaction) method at 600°C with particle size ranging between 40-60 nm and a decrease in the gelation time. he formation of apatite layer over the sol-gel prepared glass was faster than in melting bioglass ater immersion in simulated body luid. Cell viability by MTT assay conirmed the efectiveness of the prepared bioactive glass nanopowder SG-B as a bone replacement material.

5. Siqueira R.L, Peitl O, Zanotto ED (2011) Gel-derived SiO2-CaO0Na2O-P2O5 bioactive powders: synthesis and in vitro bioactivity. Mat. Sci. and Eng. C 31:983-991. 6. Radin S, Ducheyne P, Rothman B, Conti A (1997) The effect of in vitro modeling conditions on the surface reactions of bioactive glass. J Biomed Mater Res 37: 363-375. 7. Oudadesse H, Dietrich E, Gal YL, Pellen P, Bureau B, et al. (2011) Apatite forming ability and cytocompatibility of pure and Zn-doped bioactive glasses. Biomed Mater 6: 035006. 8. Dietrich E, Oudadesse H, Lucas-Girot A, Mami M (2009) In vitro bioactivity of melt-derived glass 46S6 doped with magnesium. J Biomed Mater Res A 88: 1087-1096. 9. Höland W, Vogel W, Naumann K, Gummel J (1985) Interface reactions between machinable bioactive glass-ceramics and bone. J Biomed Mater Res 19: 303-312. 10. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R (2001) Enhanced osteoclast-like cell functions on nanophase ceramics. Biomaterials 22: 1327-1333. 11. Brinker CJ, Scherer GW (1990) Sol–gel Science. Academic Press, Inc.

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